Abstract

In this report, we have assessed the role of IFN-γ as a sensitizing
agent in apoptosis mediated by activation of death receptor CD95 in
breast tumor cells. Treatment of the tumor cell lines MCF-7 and
MDA-MB231 with IFN-γ significantly facilitated apoptosis induced by
CD95 receptor ligation at the plasma membrane, independently of
p53 status. In contrast, IFN-γ treatment did not enhance the
apoptotic effect of the DNA-damaging drug, doxorubicin. Analysis of
apoptosis regulators indicated that caspase-8 mRNA and protein levels
were up-regulated in both of the cell lines after treatment with
IFN-γ. Furthermore, IFN-γ sensitized MCF-7 and MDA-MB231 cells to
CD95-mediated activation of caspase-8, induction of cytochrome
c release from mitochondria, and processing of
caspase-9. Release of cytochrome c, caspases activation,
and apoptosis were prevented in MCF-7 cells overexpressing Bcl-2.
Altogether these results indicate that IFN-γ, maybe through the
elevation of caspase-8 levels, sensitizes human breast tumor cells to a
death receptor-mediated, mitochondria-operated pathway of apoptosis.

INTRODUCTION

Apoptotic cell death plays a fundamental role in normal
development, tissue homeostasis, and pathological situations
(1,
2,
3)
. The CD95 (Fas/Apo-1) receptor, a member of the
TNF
3
/nerve growth factor receptor family
(4, 5)
, is a
potent inducer of apoptosis in the immune system on interaction with
its natural ligand CD95L, a type II integral-membrane protein
homologous to TNF-α
(6)
. Whereas the expression of CD95L
seems to be more restricted to lymphoid cells
(6)
, CD95
antigen is also expressed outside the immune system in many
nontransformed cells, including mammary epithelial cells
(7)
. In contrast, breast tumor cells express low levels of
CD95 mRNA and protein and are usually not killed by CD95 antibodies
(8)
.

IFNs are a family of natural glycoproteins that share antiviral,
immunomodulatory, and antiproliferative effects
(9)
. Their
antitumor activity against a variety of tumor cells such as lymphomas,
melanomas, and multiple myeloma has also been reported
(10, 11)
. Clinically and experimentally, it has been
demonstrated that IFN-γ can enhance the antitumor effects of
antimetabolite on cancer cells
(11, 12)
. In
vitro, IFN-γ can induce or modulate apoptosis either as a single
agent or in combination with other chemotherapeutic agents
(13)
. In breast cancer cells, positive results have been
obtained by immunotherapy with natural IFNs and interleukins,
particularly in combination strategies
(14)
. However, the
mechanism of IFNs-mediated modulation of cellular susceptibility to
apoptosis has not been elucidated. IFN-γ and IFN-α can up-regulate
the expression of a number of apoptosis-related proteins including
TNF-R, CD95 and other death receptors as well as their respective
ligands, different members of the Bcl-2 family, and caspases in
different types of cells
(13, 15, 16)
. Moreover, the tumor
suppressor IRF-1 has been proposed to play a role in apoptosis and to
be a transcriptional activator of the ICE/caspase-1 gene
(17)
. In breast cancer cells, it has been reported that
IFN-γ induces sensitization to CD95-mediated apoptosis by
up-regulating the expression of ICE/caspase-1
(8)
.
However, recent results have indicated that caspase-1/ICE does not play
a crucial role in apoptosis on death-receptor cross-linking by ligands
(18,
19,
20)
.

We have recently reported that DNA-damaging drugs sensitize breast
tumor cells to CD95-mediated apoptosis by inducing the expression of
cell membrane CD95 receptor
(21)
. The accumulation of p53
in drug-treated cells is required for the up-regulation of CD95.
However, inactivating p53 mutations are frequently observed
in breast cancer cells that abrogate p53-dependent gene transcription
(22,
23,
24)
. The above data prompted us to investigate the
effects of IFN-γ on CD95 receptor-induced apoptosis in both p53wild-type and p53 mutated breast tumor cells. In this
report, we show that IFN-γ sensitizes breast tumor cells to CD95
receptor-mediated apoptosis in a p53-independent way. Although IFN-γ
induces the expression of CD95 in the membrane of breast cancer cells,
it does not seem to be the sole mechanism by which IFN-γ regulates
apoptosis induced by death receptor ligation. We have observed a marked
up-regulation of caspase-8, which plays a pivotal role in the
proteolytic cascade leading to apoptosis on activation of death
receptors
(25,
26,
27)
. In this report, we also show that
IFN-γ facilitates the induction, on CD95 ligation, of biochemical
events such as the activation of initiator caspase-8, the release of
cytochrome c from mitochondria, and the processing of
caspase-9, which are important events in CD95-mediated apoptosis of
certain cells
(28)
. Finally, we demonstrate that apoptosis
induced by the combination of IFN-γ and a CD95 agonistic antibody
probably involves mitochondrial events, inasmuch as it is inhibited in
cells overexpressing Bcl-2.

Cell Lines.

The human breast tumor cell lines MCF-7 and MDA-MB231 were kindly
provided by Dr. M. Ruiz de Almodovar (Department of Radiology,
University of Granada, Granada, Spain). They were maintained in
culture in RPMI 1640 containing 10% fetal bovine serum, 1
mml-glutamine and gentamicin, at 37°C in a
humidified 5% CO2/95% air incubator. Stable
cell lines overexpressing human Bcl-2 protein were generated by
transfection of MCF-7 cells with either pcDNA3 or pcDNA3-hbcl-2 DNA,
using FUGENE reagent (Roche Molecular Biochemicals) according to the
manufacturer’s instructions. Resistant clones were selected in culture
medium with 2 mg/ml G418 sulfate (Sigma Chemical Co.) and analyzed for
the overexpression of hBcl-2 by Western blot.

Analysis of Cell Viability and Apoptosis.

Cell viability was determined by the crystal violet method as described
previously
(29)
. PS exposure on the surface of apoptotic
cells was detected by flow cytometry after staining with Anexin-V-FLUOS
(Roche Molecular Biochemicals). Flow cytometry was performed on a
FACScan cytometer using the Cell Quest software (Becton Dickinson,
Mountain View, CA).

For measurements of cytochrome c release, cells were
lysed and cytosolic fractions were separated from mitochondria as
described previously
(31)
. Cytosolic proteins (40 μg of
protein) were mixed with Laemmli buffer and resolved on SDS-12% PAGE
minigels. Cytochrome c was determined by Western blot
analysis as described above.

RT-PCR.

Total RNA was isolated from cells with Trizol reagent (Life
Technologies, Inc. Grand Island, NY) as recommended by the supplier.
cDNAs were synthesized from 2 μg of total RNA using a RNA PCR kit
(Perkin-Elmer) with the supplied oligo d(T) primer under conditions
described by the manufacturer. PCR reactions were performed using the
following primers: human CD95L sense, 5′-CAGGACTGAGAAGAAGTAAAACCG-3′,
and human CD95L antisense, 5′-CTCCAAAGATGATGCTGTG-3′; human Bak
sense, 5′-CCTGTTTGAGAGTGGCATC-3′, and human Bak antisense,
5′-TCGTACCACAAACTGGCCCA-3′; human IRF-1 sense,
5′-CTTAAGAACCAGGCAACCTCTGCCTTC-3′, and human IRF-1 antisense,
5′-GATATCTGGCAGGGAGTTCATG-3′; and human β-actin sense,
5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′, and human β-actin
antisense, 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′—giving products of
440, 266, 406, and 661 bp, respectively. Cycle conditions for all of
the PCR reactions were 1 min at 95°C, 1 min at 55°C, and 1
min at 72°C for 30 cycles with the exception of hCD95L. For hCD95L,
PCR was carried out under the same conditions for 40 cycles.

Northern Blot Analysis of Caspase-8 mRNA.

Total RNA (20 μg) was run on 1% agarose/formaldehyde gel and
transferred to nylon membranes (Hybond-N, Amersham Pharmacia Biotech,
Buckinghamshire, England). Membranes were hybridized to a cDNA probe
for caspase-8 labeled with [α-32P]dCTP
(Amersham Pharmacia Biotech), using a random primer labeling kit (Roche
Molecular Biochemicals). Caspase-8 cDNA probe was generated by RT-PCR,
as described above, using the following primers for PCR amplification:
sense 5′-GATATTGGGGAACAACTGGAC-3′ and antisense
5′-CATGTCATCATCCAGTTTGCA-3′.

RESULTS

Sensitization by IFN-γ of Breast Tumor Cells to Death
Receptor-mediated Apoptosis Is Independent of p53 Status.

We reported recently that breast tumor cells expressing wild type p53
can be sensitized to CD95-mediated apoptosis by treatments that caused
DNA damage
(21)
. These treatments up-regulated the cell
membrane expression of CD95 through a p53-dependent mechanism. However,
inactivating p53 mutations are frequently observed in breast
tumor cells
(24)
and can abrogate p53-dependent elevation
of cell cycle inhibitors and apoptosis-inducing molecules
(32, 33)
. To circumvent this problem, we have analyzed the ability of
IFN-γ treatment to sensitize breast tumor cells to death
receptor-mediated apoptosis in both p53 wild-type and
p53 mutated breast cancer cell lines. Treatment of both
MCF-7 (p53 wt) and MDA-MB231 (mutant p53) breast
tumor cells with the combination of IFN-γ and CD95 antibody caused an
important decrease in cell viability as determined by the crystal
violet method (Fig. 1, A and B)
⇓
. In both cell lines, treatment with
either IFN-γ or CD95 antibody had only a slight effect on the
number of viable cells at the end of the incubation period. Similar
results were obtained in EVSA-T cells, another breast tumor cell line
harboring a mutant p53 (results not shown). Analysis of PS
exposure in the extracellular side of the plasma membrane, a marker of
apoptosis, indicated that the synergistic loss of cell viability
observed (Fig. 1A)
⇓
was in fact attributable to facilitation
of this type of cell death (Fig. 1, C and D)
⇓
.
Fig. 1D⇓
also shows that synergism with IFN-γ was observed
not only with CD95 antibody but also with soluble recombinant CD95
ligand. Results not shown demonstrated that IFN-γ also sensitized
both of the human breast tumor cell lines to apoptosis induced by
TRAIL, another member of the TNF-α family of death-inducing ligands
that preferentially kills tumor cells
(34, 35)
. However,
IFN-γ did not enhance the effect of doxorubicin, a DNA-damaging drug
that at certain concentrations may cause apoptosis in breast tumor
cells (Fig. 2
⇓
; Ref.
21
).

IFN-γ sensitized breast tumor cells to CD95-mediated
cell death. Cell viability was assessed by crystal violet staining
after 48 h treatment of MCF-7 cells (A) and 72 h treatment of MDA-MB231 cells (B), without or with 10
ng/ml IFN-γ in the presence or in the absence of 500ng/ml CD95 mAb,
CH11. PS externalization in MCF-7 cells was determined after 48-h
incubation without or with 10 ng/ml IFN-γ in the presence or in the
absence of either 500 ng/ml CH11 (C) or 100 ng/ml
recombinant human CD95 ligand and 200 ng/ml CD95 ligand cross-linker
(D). Bars, SD from at least two
independent experiments.

IFN-γ did not sensitize breast tumor cells to
doxorubicin-induced cell death. Cell viability was assessed by the
crystal violet method after 48-h incubation of MCF-7 cells with
different concentrations of doxorubicin (Doxo) or CD95
antibody (500 ng/ml) in the presence of IFN-γ (10 ng/ml).
Bars, SD from two independent experiments.

Up-Regulation of CD95 Receptors by IFN-γ Is Not Sufficient to
Sensitize Breast Tumor Cells to CD95-mediated Apoptosis.

Our findings (Figs. 1
⇓
and 2)
⇓
indicated that IFN-γ enhanced the
sensitivity of breast tumor cells to CD95 receptor-induced apoptosis,
only in the presence of exogenous CD95 antibody or ligand. These
observations suggested that IFN-γ treatment was not able to induce
the expression of endogenous CD95L. This hypothesis was confirmed by
RT-PCR analysis of CD95L mRNA. Treatment with IFN-γ for up to 48 h did not induce CD95L mRNA expression in MCF-7 cells (Fig. 3)
⇓
. As a control of CD95L expression, human Jurkat T-lymphocytes, treated
in parallel for 5 h with calcium ionophore and phorbol ester,
showed a marked up-regulation of CD95L mRNA. To investigate the
mechanism underlying the sensitization of breast tumor cells by IFN-γ
to apoptosis-mediated-by-death-receptor activation, we first analyzed
the expression of CD95 receptors in cells treated with IFN-γ (Fig. 4)
⇓
. Total CD95 protein (Fig. 4A)
⇓
and CD95 receptor expression
at the cell membrane (Fig. 4B)
⇓
significantly increased after
treatment of MCF-7 cells with IFN-γ, as previously reported by other
investigators who analyzed levels of mRNA for CD95
(8)
. In
contrast, untreated MDA-MB231 cells expressed a significant amount of
total CD95 protein and CD95 membrane receptors and IFN-γ
treatment only slightly enhanced these levels (Fig. 4, A and B)
⇓
but markedly sensitized these cells to
CD95-mediated cell death (Fig. 1B)
⇓
. To further assess the
importance of the increase in CD95 levels in the facilitation by
IFN-γ of CD95-mediated cell death, we carried out several experiments
in MCF-7 cells with the DNA-damaging drug, doxorubicin. We have
previously shown that treatment of MCF-7 cells with doxorubicin
concentrations of 100 ng/ml or higher caused p53 accumulation that was
followed by a marked elevation of CD95 expression and that synergized
with CD95 agonistic antibody in the induction of apoptosis
(21)
. At a lower concentration (10 ng/ml),
doxorubicin produced an effect on CD95 expression (Fig. 4C)
⇓
that was similar to the effect observed in cells incubated in the
presence of IFN-γ (Fig. 4A)
⇓
. However, in contrast to the
sensitization to apoptosis induced by IFN-γ (Fig. 1)
⇓
, doxorubicin did
not facilitate CD95-mediated cell death (Fig. 4D)
⇓
. Although
we cannot completely exclude a certain role of increased CD95 in
IFN-γ-induced sensitization to apoptosis, these observations and the
results obtained in MDA-MB231 cells suggested that elevation of
CD95 expression by IFN-γ is not sufficient to explain the synergism
found in CD95-mediated apoptosis. The observations and results also
suggested that changes in intracellular apoptotic mediators should be
involved in the proapoptotic effect of IFN-γ in breast tumor cells.

Expression of CD95L is not induced by IFN-γ in breast
tumor cells. MCF-7 cells were treated with IFN-γ (10 ng/ml) for the
times indicated. As a control of CD95L expression, Jurkat cells
were treated with calcium ionophore A23418 and phorbol-12,13-dibutyrate
(I+P) for 5 h. After treatment, mRNA was extracted
from cells and analyzed by RT-PCR as indicated in “Materials and
Methods.”

Treatment of breast tumor cells with either IFN-γ or
doxorubicin induced similar up-regulation of CD95 expression but
differentially sensitized MCF-7 cells to CD95-induced apoptosis. CD95
protein expression was determined by Western blotting
(A) and by flow cytometry (B) in MCF-7
and MDA-MB231 cells, after treatment without or with 10 ng/ml IFN-γ
for either 24 h (A) or 48 h
(B). (C) CD95 receptors expression was
analyzed by flow cytometry in MCF-7 cells after incubation for 48 h without or with 10 ng/ml doxorubicin. (D) MCF-7 cells
were treated without or with 10 ng/ml doxorubicin in the presence or in
the absence of 500 ng/ml CD95 antibody CH11. Cell viability was
assessed after 48 h by crystal violet staining.
Bars, SD from two different experiments.

Up-Regulation of Caspase-8/FLICE in Breast Tumor Cells after
Treatment with IFN-γ.

It has been reported that IFN-γ modulates apoptosis in colon cancer
cells by sensitizing the cells to killing by apoptotic stimuli
(13)
. In these cells, IFN-γ induced changes in an array
of genes of the apoptotic pathway, including death receptors, caspases,
and the proapoptotic member of the bcl-2 family, Bak. To further
investigate the mechanism of IFN-γ-mediated facilitation of apoptosis
in breast tumor cells, we carried out the analysis of the expression of
different apoptosis-related molecules on treatment with a sensitizing
concentration of IFN-γ. In these experiments, we did not observe any
changes in the levels of the bcl-2 family proteins Bax, Bad, Bcl-2, or
Bid after treatment with IFN-γ, as determined by Western blot
analysis (Fig. 5A)
⇓
. We also determined by RT-PCR the mRNA expression for
proapoptotic Bak, the levels of which are elevated in human colon
adenocarcinoma cells after IFN-γ treatment. As shown in Fig. 5A⇓
, Bak mRNA levels remained unchanged after 36 h of
incubation in the presence of IFN-γ. In these cells, IFN-γ
treatment caused an induction of mRNA for the transcriptional activator
IRF-1, an IFN inducible gene that served as a control for IFN-γ
action
(36)
.

IFN-γ up-regulated the expression of caspase-8 in breast
cancer cells. MCF-7 and MDA-MB231 cells were treated without or with 10
ng/ml IFN-γ for the indicated periods of time. Expression of Bax,
Bad, Bcl-2, and Bid (A), and of caspase-8, FADD, and
caspase-9 (B) were determined by Western blotting. In
(A) the expression of Bak mRNA was analyzed by RT-PCR.
RT-PCR products of β-actin and IRF-1 were used as controls of RNA
input and IFN-γ action, respectively. C, expression
levels of caspase-8 mRNA were detected by Northern blot analysis in
MCF-7 and MDA-MB231 cells after treatment without or with IFN-γ (10
ng/ml) for 15 h or 36 h, respectively; lower
panel, ethidium bromide staining of loaded RNA.

Caspases, cysteine proteases of the CED3/ICE family, are
essential elements of the death receptor-initiated pathway of
apoptosis. They play a role in both the initiation and the execution
phases of apoptosis induced on death receptor cross-linking by specific
ligands
(37,
38,
39)
. It has been shown that IFN-γ can
up-regulate the expression of several members of this family in
different cells
(13, 40)
. In breast tumor cells, it was
reported that IFN-γ treatment increased the expression of
caspase-1/ICE
(8)
. It was suggested that this effect could
have a role in the IFN-γ-induced sensitization of these tumor cells
to CD95-mediated apoptosis. However, it is now clear that caspase-1/ICE
is not involved in the proteolytic cascade leading to apoptosis on
death-receptor cross-linking by ligands
(18,
19,
20)
. Instead,
ligation of death receptors results in the formation of the DISC, a
complex that comprises the adapter molecule FADD/MORT1 and caspase-8
(41)
. Formation of the DISC results in the release of
active caspase-8 and the induction of apoptosis through either a
caspases cascade (CD95 type I cells) or a mitochondria-mediated pathway
(CD95 type II cells;
28
). We, therefore, decided to
determine the levels of both FADD/MORT1 and caspase-8 in breast tumor
cells treated with IFN-γ. Results shown in Fig. 5B⇓
indicate that there was a clear up-regulation of caspase-8 protein in
both MCF-7 and MDA-MB231 cells treated with IFN-γ. In agreement with
these data, analysis of mRNA levels for caspase-8 during IFN-γ
treatment revealed a marked increase of this mRNA in both of the tumor
cell lines (Fig. 5C)
⇓
. Interestingly, it has been reported
that overexpression of caspase-8 is sufficient to sensitize cells to
apoptosis
(25)
. We also analyzed the expression of FADD
protein, the adapter molecule that recruits caspase-8 to the DISC in
death receptor-mediated apoptosis. As shown in Fig. 5B⇓
, FADD
levels did not change in MCF-7 cells incubated in the presence of
IFN-γ. Recruitment and activation of caspase-8 at the DISC can be
sometimes prevented by the presence of the endogenous inhibitor c-FLIP,
a determinant of susceptibility to death receptor-mediated apoptosis
(42)
. This protein could be highly expressed in tumor
cells
(43)
. To ascertain whether IFN-γ was reducing the
expression of such an inhibitor in breast tumor cells, we examined by
RT-PCR analysis the mRNA expression of c-FLIP in MCF-7 and MDA-MB231
cells. Results not shown indicated that these tumor cells did not
express detectable levels of c-FLIP.

Caspase-9 forms a multiprotein complex with Apaf-1 and cytochrome
c and is a key element in mitochondria-mediated caspase
activation
(44, 45)
. In the breast tumor cell lines MCF-7
and MDA-MB231, caspase-9 protein was expressed, but we did not observe
any significant change in the cellular levels of this caspase on
IFN-γ treatment (Fig. 5B)
⇓
.

IFN-γ Promotes, in Breast Tumor Cells, the Activation of an
CD95-induced Mitochondria-operated Apoptotic Pathway That Is Inhibited
by Bcl-2 Overexpression.

Mitochondria can play a pivotal role in apoptosis induced by different
apoptotic inducers, particularly DNA-damaging agents
(46, 47)
. In CD95-expressing type II cells, release of apoptotic
factors from mitochondria on CD95 activation in the plasma membrane is
a necessary step in CD95-induced apoptosis
(28)
. MCF-7
cells have been ascribed to the type II group of cells based on the
fact that Bcl-xL blocked apoptosis in cells
transfected with CD95
(28, 48)
. To get further insight
into the mechanism of IFN-γ-induced sensitization of breast tumor
cells to death receptor-mediated apoptosis, we analyzed several
biochemical events which are known to be elicited on CD95 ligation in
the plasma membrane. In this respect, CD95-mediated caspase-8
activation, which was not observed in cells treated with CD95 antibody
alone, was clearly induced by CD95 antibody in both breast tumor cell
lines when IFN-γ was present in the culture medium (Fig. 6A and B)
⇓
. Cytochrome c release from
mitochondria is a crucial step in the formation of the apoptosome,
during activation of caspase-9
(49, 50)
. Data shown in
Fig. 6, A and B⇓
, indicate that IFN-γ treatment
facilitated CD95-induced release of cytochrome c in breast
tumor cells. As a consequence probably of cytochrome c
elevation in the cytosol, there was an activation of caspase-9
processing, measured as formation of the
Mr 32,000 fragment, in cells
incubated in the presence of both CD95 antibody and IFN-γ (Fig. 6, A and B)
⇓
. It is interesting that both the
activation of caspase-8 and the release of cytochrome c can
be observed as early as 6 h after the addition of CD95 antibody,
whereas caspase-9 processing is only observed at later times (Fig. 6, A and B)
⇓
, which suggests an ordered relationship
between these events. Finally, the triggering of this apoptotic pathway
by the combination of CD95 antibody and IFN-γ resulted in the
activation of executioner caspases as determined by the proteolytic
cleavage of the nuclear substrate PARP (Fig. 6C)
⇓
.
Altogether, these results supported the hypothesis that in breast tumor
cells, IFN-γ-promoted sensitization to death receptor-induced
apoptosis was associated with the activation of a mitochondria-operated
apoptotic pathway.

IFN-γ facilitated CD95-induced activation of a
mitochondria-operated apoptotic pathway in breast cancer cells. MCF-7
(A, C) or MDA-MB231 (B,
C) cells were preincubated for 24 h in the presence
or absence of 10 ng/ml IFN-γ before treatment without or with 500
ng/ml CD95 antibody CH11 for the indicated times. In B,
middle and lower panels, IFN-γ-treated
MDA-MB231 cells were incubated with CD95 antibody for 36 h.
Activation of caspase-8, release of cytochrome c from
mitochondria, activation of caspase-9, and cleavage of PARP were
determined by Western blot. Arrows, the cleaved
intermediate forms of caspase-8 (41–43 kDa), caspase-9
(32 kDa) and the proteolytic fragment of PARP (85
kDa). In B, α-tubulin was used as control of loaded protein.
kDa, Mr in thousands.

The importance of mitochondria-regulated events in IFN-γ-induced
sensitization was further analyzed in MCF-7 cells transfected with a
cDNA encoding antiapoptotic human Bcl-2. Several clones were selected
that overexpressed Bcl-2 protein. Results obtained with a
representative clone are shown in Fig. 7
⇓
. In MCF-7bcl-2 cells, Bcl-2 protein expression
was markedly elevated as compared with cells transfected with an empty
vector (Fig. 7A)
⇓
. Interestingly,
MCF-7bcl-2 cells were completely protected from
cell death induced by the combination of IFN-γ and agonistic CD95
antibody (Fig. 7B)
⇓
. Antiapoptotic Bcl-2 inhibits the
activation of executioner caspases and apoptosis by preventing the
release of cytochrome c from mitochondria
(51)
.
As shown in Fig. 7C⇓
, both the release of cytochrome
c from mitochondria and the activation of PARP cleavage were
clearly prevented in MCF-7 cells overexpressing Bcl-2. It has been
demonstrated that in MCF-7 cells—stably expressing CD95—caspase-8 is
recruited to the DISC after the triggering of CD95
(48)
. In these cells, overexpression of
Bcl-xL did not interfere with activation
of caspase-8, but it blocked CD95-mediated apoptosis
(28, 52, 53)
. We have shown (Fig. 7, B and C)
⇓
that
overexpression of Bcl-2 prevented apoptosis as well as cytochrome
c release and the activation of executioner caspases.
However, when examining caspase-8 activation, we found no differences
between control-transfected and Bcl-2-overexpressing MCF-7 cells on
treatment with IFN-γ and CD95 antibody (Fig. 7D)
⇓
.
Taken together, our data suggest that, in IFN-γ sensitized cells,
Bcl-2 blocks the CD95 pathway downstream of caspase-8 activation and
upstream of cytochrome c release and executioner caspases.
These results also indicate that sensitization by IFN-γ to
CD95-induced apoptosis in MCF-7 breast tumor cells occurs through a
mitochondria-mediated pathway. It is interesting that in
Bcl-2-overexpressing MCF-7 cells, apoptosis was inhibited despite
activation of caspase-8 (Fig. 7
⇓
, B, C, and
D), which, in principle, could lead to activation of a
caspases cascade (type I CD95 cells). However, MCF-7 cells are
deficient in caspase-3 expression, and this caspase is required to
activate the type I pathway in MCF-7 cells that overexpress CD95
(28)
. On the other hand, it is possible that the
activation of caspase-8 that is observed in our experiments on
treatment with IFN-γ and CD95 antibody (Fig. 6, A⇓
and
B, and Fig. 7D⇓
) is not sufficient to activate the
CD95 type I pathway, in contrast to other studies using
CD95-overexpressing cells
(28, 52, 53)
, in which the
recruitment of FADD and caspase-8 to the DISC and the activation of
caspase-8 were markedly stimulated on CD95 ligation.

Overexpression of Bcl-2 prevented IFN-γ-promoted
CD95-mediated apoptosis in breast tumor cells. MCF-7 cells were
transfected with either pcDNA3-neo vector or pcDNA3-bcl-2 vector, and
Bcl-2 expression levels were determined by Western blot analysis
(A). In B, pcDNA3-neo
(control) or pcDNA3-bcl-2 (Bcl-2) cells
were treated with or without IFN-γ (10 ng/ml) in the presence or
absence of CD95 antibody (500 ng/ml) for 48 h, and cell viability
was assessed by crystal violet staining. Bars,
SD from two different experiments. Release of cytochrome
c from mitochondria and PARP cleavage (C)
and caspase-8 activation (D) were determined by Western
blot analysis in both MCF-7 and MCF-7Bcl-2 cells incubated
for 24 h with or without IFN-γ (10 ng/ml) and subsequently
treated in the presence or absence of CD95 antibody (500 ng/ml) for
24 h.

DISCUSSION

Different mechanisms seem to be involved in the resistance of
tumor cells to CD95L-induced apoptosis. Loss of CD95 receptor
expression has been observed in different tumor cells, including
hepatocellular and breast carcinomas
(7, 8, 54)
. However,
in other cases, the resistance to death receptor-mediated apoptosis is
found in tumor cells expressing a significant number of CD95 receptors,
e.g., in certain colon carcinoma cells
(55)
. In
the breast, the presence of CD95 protein in the cell surface of normal
mammary epithelial cells has been reported
(7)
. Although a
role has not been yet ascribed to CD95 in apoptosis of these cells
in vivo, normal breast epithelial cells are sensitive to
CD95-mediated apoptosis in vitro(8)
. In
contrast, malignant breast cell lines express low levels of CD95
protein and are resistant to CD95-mediated apoptosis
(8)
.
We have previously confirmed these results and demonstrated that
genotoxic agents up-regulated the expression of CD95 in breast tumor
cells by a p53-dependent mechanism and sensitized these cells to
CD95-induced apoptosis
(21)
. However, p53 is frequently
mutated and inactivated in breast tumor cells
(56)
; and,
therefore, it could be important to find alternative treatments to
sensitize malignant breast epithelial cells to death receptor-mediated
cell death. In our study, we have found that IFN-γ sensitizes breast
tumor cells to death receptor-mediated apoptosis but not to
doxorubicin-induced apoptosis. In this respect, breast tumor cells seem
to behave differently from colon cancer cells, in which IFN-γ
treatment sensitizes these tumor cells to apoptosis that is induced by
CD95 receptor activation, irradiation, and antitumor agents
(13)
. Our results also indicate that, besides its effect
on death receptor expression, IFN-γ might be regulating the
intracellular apoptotic machinery
(46)
. In this respect,
it has been reported that IFN-γ modulates a p53-independent apoptotic
pathway through the regulation of several apoptosis-related genes, in a
human colon adenocarcinoma cell line
(13)
. These genes
included CD95 and TNFR1, several members of the
caspase family, and two members of the bcl-2 family: bak and
Mcl-1. However, the relative contribution of each of these
genes to IFN-γ-mediated promotion of apoptosis was not
established in these studies.

Apart from caspase-8, no other changes were observed in the levels of
the apoptosis-related mRNAs and proteins analyzed in our study, in
contrast to colon adenocarcinoma cells
(13)
. In our
report, we have provided evidence for the up-regulation of caspase-8
mRNA and protein on IFN-γ treatment in both wild-type and mutant
p53-expressing human breast cancer cells. This caspase is the
first caspase required in death receptor-mediated apoptosis
(57)
, although it could be also activated downstream of
mitochondria through an amplification pathway regulated by this
organelle
(58)
. Caspase-8 is recruited in zymogen form to
the DISC on ligation of CD95 at the cell surface, by either CD95L or
agonistic CD95 antibodies
(25)
. After recruitment,
caspase-8 is autoprocessed to generate the active form that can cleave
other substrates, including executioner caspases. According to the
induced-proximity model for caspase-8 activation, a locally high
concentration of this caspase zymogen would promote the autoprocessing
and the release of the active caspase
(39)
. It is possible
that the increased expression of caspase-8 found in IFN-γ-treated
breast tumor cells, might facilitate formation of the DISC triggered on
CD95-receptor activation and thus subsequently activate an apoptotic
program. In this respect, it is interesting to mention that IFN-γ did
not increase the apoptotic effect of the DNA-damaging drug,
doxorubicin, in breast tumor cells. DNA-damaging treatments usually
activate apoptosis through a mitochondrial pathway that shares several
elements with death receptor-induced apoptotic mechanism in CD95 type
II cells
(59)
. However, an important difference between
both mechanisms is the absolute requirement for caspase-8 recruitment
to the DISC in death receptor-mediated apoptosis but not in DNA
damage-induced cell death
(57, 59)
. The fact that IFN-γ
sensitizes breast tumor cells to death receptor-mediated apoptosis but
not to doxorubicin-induced death, suggests that IFN-γ must be acting
at an early step in the apoptotic process, such as the activation of
initiator caspase-8 at the DISC. In CD95 type II cells like the MCF-7
cell line, mitochondria may function as amplifiers activating caspase-9
and executioner caspases
(58)
. This proposition is in
agreement with our results indicating the activation by CD95 agonistic
antibody of a mitochondria-regulated pathway of apoptosis in
IFN-γ-treated cells. The importance of this IFN-γ-promoted
mitochondrial pathway was confirmed by experiments in breast tumor
cells that overexpressed Bcl-2. In these cells, the release of
cytochrome c from mitochondria, the activation of
executioner caspases, and apoptosis were markedly inhibited.

Previous data
(8)
have indicated that caspase-1/ICE is
up-regulated in some breast cancer cell lines on treatment with
IFN-γ. This study also showed that overexpression of caspase-1/ICE
sensitized these cells to CD95-mediated apoptosis. However, more recent
data have demonstrated that caspase-1/ICE is not involved in the
proteolytic cascade activated on CD95 cross-linking at the cell surface
by CD95L or CD95 antibody
(18,
19,
20)
. An explanation for the
observed increase in CD95-mediated apoptosis in
caspase-1/ICE-transfected MCF-7 cells
(8)
is that
overexpression of this caspase somehow replaced caspase-8 in the
activation of the apoptotic machinery
(60)
. Alternatively,
overexpressed caspase-1/ICE could be a substrate of CD95-activated
caspase-8 and provoke an amplification of caspase signaling
(61)
. How IFN-γ can regulate the expression of the
caspase-8 gene is not known. IFN-γ activates the signal transducer
and activator of transcription (STAT) signaling pathway, which
can play important roles in cell proliferation, differentiation, and
apoptosis
(9, 62)
. Furthermore, it was demonstrated that
IFN-γ activated STAT1 and induced apoptosis in various cell
types
(63)
. Activation of apoptosis by IFN-γ
correlated with the induction of ICE/caspase-1
(63)
.
IFN-γ-induced activation of the caspase-1 promoter was dependent on
the binding of IRF-1
(64)
. Although the elements in the
caspase-8 gene that are responsible for activating caspase-8
expression have not been identified, one can speculate with the
possibility of similarities between caspase-1 and -8 in terms of the
mechanism regulating their expression by IFN-γ.

Regulation of the expression and/or activity of the DISC components
could be a strategy used by virally infected or tumor cells to escape
from the host immune system. Protection of virus-infected cells against
death-receptor-induced apoptosis may lead to higher virus production
and contribute to the persistence and oncogenicity of several
FLIP-encoding viruses
(65)
. In this respect, caspase-8
inhibitors like CrmA and v-FLIP are present in cells infected by
different viruses
(43, 66)
. Down-regulation of CD95
expression is also observed in adenovirus-infected cells
(67)
. Human melanomas express elevated levels of FLIP and
are resistant to death receptor-mediated apoptosis
(27)
.
The gene for caspase-8 is frequently inactivated in neuroblastoma, a
tumor of the peripheral nervous system
(68)
. Caspase-8 is
a cellular target of the Mr 14,700
protein of adenovirus type 5, that protected cells from death
receptor-induced apoptosis
(69)
. Therefore,
down-regulation of caspase-8 levels or activity in tumor cells may be
an important mechanism in the evasion of the immune response. In this
respect, our data indicate that sensitizing regimens like
IFN-γ may be used in combination strategies with nontoxic
death-receptor ligands, for instance TRAIL, in the treatment of human
breast cancer.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by grants from Fundación
Ramón Areces and Ministerio de Educación y Cultura
(1FD97-0514-C02-01) to A. L-R. C. M-P. is recipient of a
fellowship from Ministerio de Educación y Cultura.